Exocytosis underlies neurotransmitter and hormone release. In neurons, synaptic vesicles (SV) packaged with neurotransmitter fuse with the plasma membrane to release their content that is sensed across the synaptic cleft. This process is tightly regulated: release is stimulated by a local increase in the free calcium concentration following the arrival of an action potential. Hormones are released in a similar fashion using some of the same protein machinery, via fusion of hormone containing secretory granules (SG) with the plasma membrane. The initial connection between a SV or SG and the plasma membrane is a small pore (~1 nm wide) that can open and close in succession before either closing permanently (transient, or kiss-and-run fusion) or dilating fully. There is large variabiliy in behavior between cell types (pore open times span ~100 ?s to 10s of s) and within the same cell (some pores flicker, some dilate abruptly). Pore flickering is modulated by physiological inputs such as stimulation strength, with important consequences about what is released (only small cargo can escape through a small pore), on what time course, and how exocytosis is coupled to endocytosis. Despite the fundamental importance of fusion pores in regulating neurotransmitter and hormone release, very little is understood regarding mechanisms controlling pore nucleation and dynamics. This is mainly due to difficulties in studying fusion pores in reconstituted systems with well-defined protein and membrane components that would allow isolating the role of each component. Fusion mediated by exocytotic SNARE proteins and their regulators has been reconstituted and studied for the past 15 years. However, existing methods are not able to resolve single reconstituted fusion pores and follow pore dynamics with sufficient time resolution. We aim (1) to engineer novel experimental approaches to enable probing mechanisms of nucleation and flickering of exocytotic fusion pores. Combining electrophysiological methods, single-particle fluorescence, microfabricated devices, and artificial bilayer technologies we will develop in vitro assays that allow direct, simultaneous monitoring of single pore flickering and lipid mixing; counting protein numbers and/or probing protein-protein interactions; and controlling membrane curvature and tension. Using these assays, we will then (2) determine factors that govern nucleation and dynamics of SNARE- mediated fusion pores. We will resolve how membrane mechanics and the dynamics of fusion proteins together determine the number of SNARE complexes required for fusion. Further, we will quantify the roles of membrane tension, constraints mimicking the cytoskeleton, curvature, and mutations on pore flickering and expansion. These fundamental studies will advance our understanding of how neurotransmitter and hormone release are regulated, with potential impact on human health in the long term.